There are numerous industrial applications where it is highly desirable for microorganisms to multiply and/or to synthesize and excrete products, such as proteins, under conditions outside but not lethal, the normal conditions for optimum vegetative growth.
When a wide variety of organisms are exposed to different types of stress, synthesis of specific proteins are induced. Such types of stress include elevated temperature, cold-shock, ethanol, heavy metal ions, anoxia, glucose starvation and others. DNA-damaging agents like nalidixic acid, UV irradiation, and bacteriophage infection have been shown to induce all or part of the heat-shock response. Concurrently with the induction of, for instance, these heat-shock proteins, microorganism cells become more tolerant to high temperature exposure normally lethal to the microorganism.
When certain microorganisms, procaryotes like Escherichia coli, are exposed to a sudden increase in ambient temperature, a shift occurs from the synthesis of normal cellular proteins to the synthesis of what are called "heat-shock" proteins. The synthesis of these proteins after incubation at elevated but non-lethal temperature is correlated with the induction of thermo-tolerance, which is the enhanced ability of the microorganism to withstand subsequent high temperature which is normally a lethal high temperature.
Cold-induced genes and expressed proteins have been reported in E. coli, which have been termed cold-shock induced. Induction of the synthesis of several proteins in response to cold-shock (from 37.degree. C. to 10.degree. C.) has been reported in E. coli (Jones et al., 1987). A gene coding one of these cold-shock proteins was cloned and sequenced (Goldstein et al., 1990).
Heat-shock response in E. coli has also been described (Neidhardt et al., 1984). When cell cultures are transferred from 30.degree. C. to 42.degree. C. heat-shock proteins are transiently expressed. The induction has been shown to be accomplished primarily by an alternate .sigma.-subunit of RNA polymerase (.sigma..sup.32), encoded by rpoH (htpK) which recognizes specific heat-shock promoters (Grossman et al., 1987; Strauss et al., 1987). It has also been shown that E. coli responds to cold temperature. However, when growing cell cultures were transferred from 37.degree. C. to 10.degree. C., several peptides were induced, none of which were heat-shock protein. Certain proteins are induced by both cold- and heat-shock. A developmentally regulated membrane protein (Maniak and Nellen, 1988) and ubiquitin (Muller-Taubenberger et al., 1988) of Dictyostelium discoidum were shown to be induced in response to cold- and heat-shock.
This invention contributes to clarification of the scientific phenomena involved in stress-induced e.g., cold- and/or heat-induced proteins, which is of major scientific significance. Further, the stress-induced, for instance heat- and/or cold-shock proteins, can have numerous commercial applications. Such applications include the ability of growing selected cells (e.g., of microorganism) under conditions under which the cells normally would not grow, or not grow as well. This invention discloses and suggests several important industrial applications.
Yeast organisms are well suited for commercial expression of heterologous proteins, having advantages over tissue culture and bacteria. First of all, highly developed technology exists for yeast fermentation processes. Yeast can be employed safely since handling and disposal methods are well established. In addition, yeast is an eucaryotic organism which has advantages when expressing genes originating from other eucaryotic backgrounds. Most of all, yeast is very well characterized and can be manipulated genetically to maximize expression of heterologous peptides.
The ideal expression system for yeast as a microbial "factory" would allow for enhanced protein production, controlled during portions of the fermentation process. Products are currently made utilizing multi-copy plasmids, efficient promoters and amplified genes. However, these systems still lack the ideal parameter, which involves the ability to direct product synthesis at a specific time or growth conditions during the fermentation. By separating the growth process (which may involve days of culture incubation) from that of product synthesis (which may occur within hours), one can obtain higher levels of product. The product is exposed for less time in the media or within cells where it may be susceptible to proteolytic degradation, and any detrimental effect of the product on the growth of the organism is removed by largely separating the growth and production processes.
In accordance with the invention, conditions have been identified under which yeast synthesizes certain desirable proteins in high yield. These conditions relate principally to the stage of growth of the yeast and the temperature, which is outside of the normal physiologically ideal temperature conditions for vegetative growth. In accordance with the invention, genes are induced in selected microorganisms (as will be described hereinafter), preferably yeasts. Microorganisms like eucaryotes and procaryotes transformed with genes of the invention are suitable for growth at low temperatures and/or at high temperatures and produce products, like proteins which normally are not produced at all or in yields not commercially interesting.
Another interesting area is the production of functional proteins when produced by recombinant DNA or fermentation techniques. It is known that proteins are often enzymatically degraded or folded improperly at physiological temperatures resulting in the proteins' decreased physiological activity. The invention provides the possibility of growing transformed cells at temperatures at which the products would retain physiological activity. For instance, an advantage of production of the proteins at reduced temperature would be to minimize improper folding or degradation.
Thus, in accordance with one embodiment of the invention, a host cell transformed with one or more genes (or part thereof) of the invention can express a functional protein of commercial value which will be produced though the ambient temperature has been reduced or raised outside the normal optimum growth temperature. This will allow the expression of the target protein to proceed with no adverse effects on the desired physiological activity of the protein. Transformed host cells and/or the protein products can also be useful in the field of agriculture in protecting plants against frost or heat damage; for production of food products, and any other biological (or pharmaceutical) products that should be protected from a stress-creating situation like extreme temperatures.
An important practical application of the invention is the mass growing of transformed cells (e.g., eucaryotes like yeasts) at lower than normal growth optimum temperature, thus achieving substantial savings in energy, or growing the cells at higher than (or at normal) optimum growth temperatures while producing more rapidly and/or higher yield of the product desired.
Further, the transfected microorganism can be made to have higher resistance to the growth inhibitory effect of the environmental stress (e.g., alcohol) and thus grow for longer periods of time and/or produce higher yields of the desired product. There are several other practical applications described herein and others will become apparent from the teachings of the invention.
Publications that may be of interest to one skilled in the art, are listed at the end of this document.